The structure, function, and subunit interaction in the multienzyme complex tryptophan synthase are being investigated. We have determined the primary sequence of the first 99 residues of the beta 2 subunit and located 3 active site residues therein: cysteine-62, lysine-87, and histidine-86. Two other important cysteine residues have been shown to be cysteine-170 and cysteine-230. The function of tryptophan synthase has been probed by spectroscopic studies of the interaction between the cofactor, pyridoxal phosphate, and certain amino acid substrates, products, and substrate analogs. An intermediate formed during the reaction of L-serine with enzyme-bound (3H)pyridoxal phosphate has been reduced by NaBH4 and isolated. The interaction between the alpha and beta 2 subunits of tryptophan synthase has been studied by ultraviolet difference spectroscopy and by studies using limited tryptic proteolysis. Two proteolytic fragments of the alpha subunit have been isolated and shown to refold independently and reassociate to give active enzyme.